The first commercial fungicide—the "Bordeaux mixture" of copper sulfate and lime—was used to fight downy mildew in French vineyards. The fungicide worked by catalyzing the production of free radicals that damage proteins and enzymes involved in cycling copper between Cu(I) and Cu(II) oxidation states in the cellular electron transport chain. However, not all fungi are sensitive to copper toxicity. Some, called mycorrhizae, which live underground in symbiosis with host plants through intracellular or extracellular colonization of their roots, are resistant, although it is not known why. A team from the CNRS and Université Joseph Fourier in Grenoble in collaboration with researchers at the University of Illinois at Chicago and in partnership with the French company Phytorestore has discovered a new form of copper—metallic nanoparticles—in the rhizosphere (soil-root interface) that may explain how mycorrhizal (symbiotic) fungi detoxify copper.

Detoxifying Copper in Soils

Copper is essential to life, but also toxic if consumed in excess. Unfortunately, it is frequently concentrated in soils as a result of pesticide application, sewage sludge deposition, mining, smelting, and industrial activities. Because some plants can tolerate this and other heavy metals and because the human food chain begins with plants, it is critical to understand how the toleration arises. The mechanism is also crucial to phytoremediation, a green technology that relies on plants to decontaminate or contain polluted soils and sediments and to purify industrial wastewaters and water leached from landfills.

Manceau et al. have discovered, using x-ray microanalyses, that common wetlands plants growing in contaminated soil in the natural environment can transform the harmful copper into metallic nanoparticles in and near roots with assistance by certain fungi. Formation of nanometallic copper in this way removes copper from the soil water and prevents it from entering the stems and leaves of the plants. The mechanism is likely common but previously undetected, owing to difficulty in analyzing and imaging complex natural materials. While farmers (and wine enthusiasts!) appreciate that copper pesticides and fungicides are used to protect crops, here fungi are implicated in the contrary role of defending wetland plants used in phytoremediation. The far-reaching results should also be of medical interest because copper toxicity is associated with human diseases (e.g., Menke’s, Wilson’s, Alzheimer’s, prion diseases, and atherosclerosis).

Specifically, the French-led team identified and imaged a new mode of copper biomineralization in a contaminated environment that is clearly associated with soil biota—plants and fungi. Reduction of divalent copper to metallic copper nanoparticles occurred in complex, contaminated soil under oxidizing conditions during exposure to natural climate variations, including a period of an exceptional heat wave. Images show compartmentalization of metallic copper within roots and along fungal hyphae of the common wetlands plants Phragmites australis (common reed) and Iris pseudoacorus.

While previous laboratory and greenhouse experiments had shown that Au3+, Ag+, H2SeO3, Te4+, and Hg2+ can be reduced to the metallic state by plants or fungi, to address the complex relationship between fungi and plants under natural environmental and climate influences, outdoor experiments are needed. Moreover, formation of nanometallic copper in oxygenated soils is likely common but previously missed because it is difficult to analyze and image complex natural soils without altering the microstructural relationship of mycorrhizae with roots, and it is easy to alter the original speciation of copper during sample preparation and exposure to the x-ray beam. Copper reduction also requires searching for a suitably strong natural reductant to explain the presence of the copper nanoparticles.

The team imaged the distribution of copper at the soil–root interface by micro x-ray fluorescence (m-XRF), and identified the new copper species as metallic nanoparticles by micro extended x-ray absorption fine-structure (m-EXAFS) spectroscopy and micro x-ray diffraction (m-XRD) on the ALS microfocus Beamline 10.3.2. They verified the uniqueness of the microanalytical results obtained on a few small areas by analyzing the bulk EXAFS spectrum for a sample representing the entire rhizosphere recorded on the FAME beamline at the ESRF.

Top: Micro x-ray fluorescence map of the area in the optical micrograph showing the distribution of zinc (red), copper (green), and calcium (blue) in the rhizosphere (soil–root interface) of I. pseudoacorus. The longitudinal section (left) shows the association of nanoparticulate metallic copper with ramified (branched) mycorrhizal fungi; the transversal section (right) shows the association in a biofilm at the surface of the root. Bottom: enlargement of a ramified hypha (long, filamentous fungus cells). Resolution = 8 x 8mm2.

The researchers propose that the transformation of toxic copper cations to copper metal is driven by the biotic production of ascorbic acid, a well-known anti-oxidant. However, this reduction of Cu2+ to Cu0 cannot occur homogeneously in an aqueous solution and appears to be facilitated only in the presence of a templating substrate, as used in the synthesis of nanomaterials. In these processes, biomolecules, such as DNA in the presence of oxygen in the dark, are used as templates with the addition of a reductant to control the shape and size of metallic nanoparticles.

Promoting the formation of copper metal in the rhizosphere could be a highly efficient method for remediating copper contamination. In contrast to metal-hyperaccumulating plants, the soil-root zone itself could be an economic source of bio-recycled copper; moreover, rhizosphere containment would prevent copper from entering the food chain via herbivores, limiting potential risks to humans. However, improving phytoremediation technologies used to clean up metal-contaminated environments requires a more detailed characterization of the role of the symbiosis between mycorrhizal fungi and host plants. The new results should also be of medical interest because copper toxicity is associated with human diseases (e.g., Menke’s, Wilson’s, Alzheimer’s, prion diseases, and atherosclerosis).

Transversal section of a root of Phragmites australis with aggregates of copper metal nanoparticles in the cortical region. The empty cavities between the cells containing copper are an airy tissue, called aerenchyma, that facilitates oxygen flow from leaves to roots. In contrast to I. pseudoacorus, copper is mostly inside the roots but still likely associated with fungal hyphae, since roots of this plant usually are colonized by arbuscular (root penetrating) endomycorrhizae. The central (stele) region that contains vascular bundles has no copper. Resolution = 4 x 4 mm2.